This is a proposal to elucidate molecular mechanisms by which mutations in the alpha1-antitrypsin gene result in emphysema and liver disease. In conjunction with these studies we shall develop and improve methods of correcting the PiZ defect in the alpha1-antitrypsin gene at the level of genomic DNA in human cells. Alpha1-antitrypsin is a major serum protein synthesized in the liver and is the primary inhibitor of neutrophil elastase in the human lung. The PiZ defect is a mutation causing reduced secretion from the liver and leading to early-onset emphysema and liver disease in homozygous individuals. We have constructed vectors containing one or the other of two point mutations characteristic of the PiZ gene. By transfecting with these vectors we have constructed, for the first time, human liver cell lines expressing PiZ-mutated alpha1-antitrypsin genes. Using these vectors and these cell lines, we shall determine relative contributions of the two point mutations to alterations in structure and intracellular processing of the defective gene products. Using the mutated alpha1-antitrypsin genes linked to an inducible promoter, we shall determine the sequence of pathological events following induction. We shall employ a transfection method yielding high levels of homologous recombination to elucidate mechanisms through which a normal, PiM copy of the alpha1-antitrypsin gene can be targeted to supplant a mutated PiZ copy in cultured cells. We shall employ new PCR-based technology to select transfected cell lines in which a defective PiZ gene has been corrected by targeted recombination with a normal PiM gene. These studies will contribute to both understanding and treatment of the debilitating PiZ defect.